How multiple farming conditions correlate with the composition of the raw cow's milk lactic microbiome.

Questionnaires on farming conditions were retrieved from 2129 dairy farms and clustered, resulting in 106 representative raw cow's milk samples analysed in winter and summer. Substantiating the efficiency of our survey, some farming conditions affected the milk physicochemical composition. Culturing identified several species of lactic acid bacteria (LAB) per milk, whose number increased through 16S ribosomal RNA (rRNA) gene sequencing and shotgun metagenome analyses. Season, indoor versus outdoor housing, cow numbers, milk substitutes, ratio cattle/rest area, house care system during lactation, and urea and medium-chain fatty acids correlated with the overall microbiome composition and the LAB diversity within it. Shotgun metagenome detected variations in gene numbers and uniqueness per milk. LAB functional pathways differed among milk samples. Focusing on amino acid metabolisms and matching the retrieved annotated genes versus non-starter lactic acid bacteria (NSLAB) references from KEGG and corresponding to those identified, all samples had the same gene spectrum for each pathway. Conversely, gene redundancy varied among samples and agreed with NSLAB diversity. Milk samples with higher numbers of NSLAB species harboured higher number of copies per pathway, which would enable steady-state towards perturbations. Some farming conditions, which affected the microbiome richness, also correlated with the NSLAB composition and functionality.

Role prediction of Gram-negative species in the resistome of raw cow's milk.

Extended use of antibiotics in dairy farming for therapeutic and prophylactic reasons, but also the higher prevalence of antibiotic resistant bacteria (ARB) in the farm environment raised the concern of consuming raw cow's milk and its derived products. The aim of this study was to predict by shotgun metagenomic analyses the presence of antibiotic resistance genes (ARGs) mainly correlated with Gram-negative bacteria in antibiotic residue free raw cow's milk derived exclusively from healthy animal from South Tyrol (Northern Italy), chosen as a model system. Assessment of shotgun metagenomic data of reconstructed scaffolds, revealed the existence of Pseudomonas spp. as the most abundant Gram-negative species in the raw cow's milk samples bearing ARGs. Besides, ARGs also linked to lactic acid bacteria such as Lactococcus sp. and Lactobacillus sp. ARGs correlated to microbiome found in milk samples conferred resistance towards aminoglycoside-streptothricin, beta-lactamase, macrolide, tetracycline, carbapenem, cephalosporin, penam, peptide, penem, fluoroquinolone, chloramphenicol and elfamycin antibiotics. Further bioinformatic processing included de-novo reassembly of all metagenomic sequences from all milk samples in one, to reconstruct metagenome assembled genomes (MAGs), which were further used to investigate mobile genetic elements (MGE). Analyses of the reconstructed MAGs showed that, MAG 9 (Pseudomonas sp1.) contained the oriT gene (origin of transfer gene) needed for transferring virulent factors. Although the presence of Pseudomonas is common in raw cow's milk, pasteurization treatment reduces their survivability. Nevertheless, attention should be paid on Pseudomonas spp. due to their intrinsic resistance to antibiotics and their capability of transferring virulent factors to other bacteria.

Potentially probiotic or postbiotic pre-converted nitrite from celery produced by an axenic culture system with probiotic lacticaseibacilli strain.

The present study evaluated the use of the probiotic Lacticaseibacillus paracasei DTA-83 as a nitrite-reducing agent to produce potentially probiotic or postbiotic pre-converted nitrite from celery. The results obtained were compared to those achieved by direct addition of sodium nitrite for the typical reddish color formation in cooked pork sausages and the inhibitory potential against the growth of target microorganisms, including the clostridia group. Regarding the sausages color, similar findings were observed when comparing the use of pre-converted nitrite from celery produced by L. paracasei DTA-83 and the direct addition of sodium nitrite. Additionally, it presented an inhibitory effect against Salmonella spp., which was not observed with the direct addition of nitrite, revealing a potential strategy to control salmonellosis in the matrix. However, a non-equivalent preservative effect against Clostridium perfringens (INCQS 215) was determined. The results highlight a promising alternative to produce probiotic or postbiotic meat ingredients; however, further studies should be conducted to investigate doses that achieve microbial control.

New insights into the variability of lactic acid production in Lachancea thermotolerans at the phenotypic and genomic level.

Non-conventional yeasts are increasingly applied in fermented beverage industry to obtain distinctive products with improved quality. Among these yeasts, Lachancea thermotolerans has multiple features of industrial relevance, especially the production of l(+)-lactic acid (LA), useful for the biological acidification of wine and beer. Since few information is available on this peculiar activity, the current study aimed to explore the physiological and genetic variability among L. thermotolerans strains. From a strain collection, mostly isolated from wine, a huge phenotypic diversity was acknowledged and allowed the selection of a high (SOL13) and a low (COLC27) LA producer. Comparative whole-genome sequencing of these two selected strains and the type strain CBS 6340T showed a high similarity in terms of gene content and functional annotation. Notwithstanding, target gene-based analysis revealed variations between high and low producers in the key gene sequences related to LA accumulation. More in-depth investigation of the core promoters and expression analysis of the genes ldh, encoding lactate dehydrogenase, indicated the transcriptional regulation may be the principal cause behind phenotypic differences. These findings highlighted the usefulness of whole-genome sequencing coupled with expression analysis. They provided crucial genetic insights for a deeper investigation of the intraspecific variability in LA production pathway.

Volatile organic compounds from Starmerella bacillaris to control gray mold on apples and modulate cider aroma profile.

Gray mold caused by Botrytis cinerea is a fungal disease that can determine significant economic losses of apple during the storage phase. An alternative to reduce the use of traditional synthetic fungicides is to employ the yeast Starmerella bacillaris as biological control agent (BCA), also with positive effect on apple juice fermentation for the production of cider. Thus, we aimed to evaluate the safety of 16 S. bacillaris strains and their ability to control B. cinerea. In addition, the fermentation performances in apple juice and the volatile organic compounds (VOCs) profile were assessed, both in single-strain and in sequential fermentations with Saccharomyces cerevisiae. The in vitro assays showed that all S. bacillaris strains can be considered safe from the analyzed virulence factors, and were able to significantly constrain the growth of B. cinerea, reducing mycelial growth of 50% in dual-culture and of 90% through VOCs. Moreover, in vivo antagonistic assays revealed a visible decrease of gray mold rot symptoms on apples confirming the potential of S. bacillaris as BCA. GC-MS analysis of the ciders obtained showed increased concentrations in the sequential fermentation of some higher alcohols and terpenes, positively correlated with the cider aromatic quality, and suggested the involvement of benzyl alcohol, known for its antimicrobial action, in the biocontrol efficacy.

Contribution of non-Saccharomyces yeasts to wine volatile and sensory diversity: A study on Lachancea thermotolerans, Metschnikowia spp. and Starmerella bacillaris strains isolated in Italy.

Saccharomyces cerevisiae starter cultures are largely used in winemaking to repress the wild microorganisms and achieve more predictable and desired outcomes. Notwithstanding, alternative microbial resources received increasing attention for their potential to produce wines with more distinctive and typical features. Our previous survey revealed a great inter- and intra-species diversity in an extensive collection of non-Saccharomyces wine yeasts from multiple regions of Italy. This study aimed to explore the detected biodiversity evaluating the quality of wines obtained by sequential inoculation of specific selected strains of the collection (Lachancea thermotolerans or Metschnikowia spp. or Starmerella bacillaris), and S. cerevisiae EC 1118. Fermentations of natural grape must at laboratory scale were followed by microbiological, chemical and sensorial analysis of the wines. The results indicated that each yeast species and strain exerted a distinctive impact on the wine, giving final products clearly separated with Principal Component Analysis. In particular, L. thermotolerans contributed producing relevant amounts of lactic acid and had the highest potential to reduce ethanol content; the presence of S. bacillaris increased the level of glycerol, and, remarkably, reduced acetaldehyde and total SO2; Metschnikowia spp. promoted the formation of higher alcohols and esters, and reduced volatile phenols. The sensory analysis based on the orthonasal aroma confirmed the separation between the wines obtained with the sequential fermentations and the control with single inoculation of EC 1118, although the three non-Saccharomyces species used could not be clearly distinguishable by the panelists. This study indicates that the use of selected native non-Saccharomyces strains in conjunction with S. cerevisiae positively modulates some relevant chemical parameters, and improves the aromatic intensity of wine, therefore justifying investments in non-conventional yeasts as co-starter cultures.